T lymphocyte function requires the meaningful interaction of T cells with the extracellular environment. Members of the beta1 or VLA integrin subfamily mediate adhesion of cells to other cells and to extracellular matrix components; these adhesive interactions are important in a variety of biological processes, including embryogenesis and tumor growth and metastasis. In the immune system, beta1 integrins have been shown to be important to T cell activation and localization to specific anatomic sites. Two aspects of B1 integrin function that are particularly relevant to T cell function are: 1) the rapid upregulation of beta1 integrin functional activity upon T cell activation, divalent cation modifications, or triggering by activating beta1-specific mABs; and 2) tyrosine phosphorylation events initiated by interaction of beta1 integrins with ligand. A complete understanding of beta1 integrin function requires identification of the functional domains of the receptor. We have recently isolated a derivative of the Jurkat T cell line that lacks expression of the beta1 chain. Since beta1 integrin activity on Jurkat cells, like peripheral T cells, can be increased by activation or divalent cations, this mutant provides an important tool for analyzing human beta1 integrin structure and function. In Aim 1. this mutant will be used to generated a panel of transfectants that express beta1 proteins with specific cytoplasmic mutations. These transfectants will be tested for binding to fibronectin. VCAM-1, and invasin after activation with various stimuli, and binding to VCAM-1 under conditions of physiological shear flow. Engagement of beta1 integrins on the H9 T cell line by antibody or natural ligands results in rapid tyrosine phosphorylation of a 105 kd protein that is distinct from pp125 (FAK). Aims 2 and 3 will elucidate structural and functional requirements for beta1 integrin-mediated tyrosine phosphorylation in H6T cells. In Aim 2 the regions of the cytoplasmic tail critical for beta1-mediated tyrosine phosphorylation will be determined by construction and expression in H9 cells of a heterologous receptor consisting of the CD2 extracellular and transmembrane domains with mutagenized beta1 cytoplasmic tails. In Aim 3, the p105 substrate will be identified by: 1) microsequencing analysis and cloning of the p105 gene; and 2) the generation of p105-specific antibodies. These reagents will then be used to characterize the expression and function of the p105 molecule. These studies will provide critical insights into activation-dependent regulation of B1 integrin functional activity and beta1 integrin-mediated tyrosine phosphorylation in human lymphocytes. These insights will be important not only to our understanding of integrin function in general, but will also provide the detailed structural information necessary for the development of therapeutic agents that specifically modulate integrin- mediated adhesive events in vivo.